Summary Transcription activation, a key step in gene control, is the readout of many signaling pathways controlling cell growth, development and stress response and defects in activation cause many human diseases and syndromes. Activation typically results from factors containing activation domains (ADs) binding to coactivator complexes containing activator binding domains (ABDs). Most known ADs are unstructured in the absence of a binding partner and many interact with multiple and structurally-unrelated ABDs. The mechanisms used by ADs to interact with coactivators, the specificity of these interactions, and mechanisms of coactivator cooperativity are critical unanswered questions for understanding the molecular basis of gene regulation. The long-term goal of this project is to determine mechanisms used by gene-specific activators and coactivators to regulate RNA polymerase (Pol) II transcription. The objectives of this proposal are to determine: mechanisms used by ADs to recognize their coactivator targets, cooperative mechanisms used by the coactivators Mediator and TFIID, and what constitutes a functional AD and ABD. This work will utilize an interdisciplinary combination of biochemical, structural, molecular, and computational approaches to examine activation in S. cerevisiae. These mechanisms are likely to be conserved, since nearly all activators tested function in both yeast and mammalian cells. To understand these mechanisms, we will build upon several breakthrough concepts and methods developed in the past grant period. We will use protein crosslinking and mass spectrometry, combined with state-of-the-art NMR to examine mechanisms used by broadly-acting ADs when binding their coactivator targets - in both large physiologically relevant complexes and at the atomic level. We will examine cooperativity between Mediator and TFIID, two conserved and widely used transcription coactivators. Finally, we will use a combination of molecular, structural, and computational approaches to delineate what comprises AD function. These new approaches will reveal mechanisms of activator binding and specificity, mechanisms of coactivator cooperation, and find rules that explain a large class of broadly acting ADs. Our proposed research is significant because it will reveal new molecular recognition mechanisms that are important for understanding transcriptional regulation as well as a wide variety of interactions involving inherently disordered proteins that function in many biological systems.